Methods to treat neurodegenerative diseases

ABSTRACT

The present invention is based on the discovery that motor neurons derived from patients with a neurodegenerative disease have decreased delayed rectifier potassium current and increased persistent sodium current compared to motor neurons derived from control healthy individuals. The present invention is also based on the discovery that the class of compounds known as “potassium channel openers” can be used to treat neurodegenerative diseases, including ALS, Parkinson&#39;s disease, Alzheimer&#39;s disease, epilepsy, and pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 U.S. National Application ofPCT/US2013/053766, filed on Aug. 6, 2013, which claims priority under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/680,662,filed on Aug. 7, 2012, and 61/791,055, filed on Mar. 15, 2013, theentire contents of which are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The claimed methods and compositions relate to methods to treatneurodegenerative diseases.

BACKGROUND

Amyotrophic lateral sclerosis (ALS) is a devastating progressiveneurodegenerative disease in which 50% of patients die within 30 monthsof onset. Some 10% of cases are familial, and mutations in copper/zincion-binding superoxide dismutase 1 (SOD1), repeat expansions in theC9orf72 gene, and mutations in fused-in-sarcoma (FUS) comprise as manyas 75% of them. C9orf72 expansions also account for approximately 15% ofsporadic ALS cases. A mouse model overexpressing human mutant SOD1 G93Arecapitulates many features of the disease, such as motor neurondegeneration and early death. However, the vast majority of ALS casesare not due to SOD1 mutations, and the relevance of animal model tothese sporadic ALS patients remains unclear. Creating human motorneurons via stem cell technology now offers an opportunity to study theproperties of human motor neurons derived from ALS patients, and therebydisease pathology, in both familial and sporadic ALS cases as well as toprovide a means to screen for drugs that may treat or reduce a risk ofdeveloping the disease.

SUMMARY

The present invention is based, in part, on the discovery that motorneurons derived from patients with a neurodegenerative disease, e.g.,ALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, or pain, havedecreased delayed rectifier potassium current and increased persistentsodium current compared to motor neurons derived from control healthyindividuals. The present invention is also based, in part, on thediscovery that the class of compounds known as “potassium channelopeners” can be used to treat neurodegenerative diseases, including ALS,Parkinson's Disease, Alzheimer's Disease, epilepsy, and pain.

Accordingly, in one aspect, the present specification provides methodsof treating or reducing a risk of developing a neurodegenerativedisease, e.g., ALS, e.g., familial ALS or sporadic ALS, Parkinson'sDisease, Alzheimer's Disease, epilepsy, or pain, in a subject. Themethods can include administering to the subject a therapeuticallyeffective amount of a potassium channel opener, thereby treating orreducing the risk of developing the neurodegenerative disease in thesubject. In some embodiments, the potassium channel opener is a KCNQ/Kv7channel opener, e.g., retigabine, e.g., a halogenated and/or afluorinated derivative of retigabine, meclofenamic acid, diclofenac, andBMS-204352. In some embodiments, the potassium channel opener is aK_(ATP) channel opener, e.g., diazoxide, minoxidil, nicorandil,pinacidil, and levcromakalim. In some embodiments, the potassium channelopener is a G protein-coupled inwardly-rectifying potassium channelopener, e.g., flupirtine. In some embodiments, the potassium channelopener is a voltage-gated Ca²⁺-activated potassium channel opener or aninward rectifier potassium channel opener. In some embodiments, themethods can further include administering to the subject ananti-neurodegenerative therapy, e.g., riluzole.

Also provided herein are methods of identifying a candidate compound totreat neurodegenerative diseases, e.g., ALS, Parkinson's Disease,Alzheimer's Disease, epilepsy, and pain. The methods can includeproviding a motor neuron from a subject with a neurodegenerativedisease, e.g., a neurodegenerative disease characterized byhyperexcitable neurons; contacting the motor neuron with a testcompound, e.g., polypeptides, small molecules, ribonucleic acids, anddeoxyribonucleic acids; determining a level of activity of the motorneuron; comparing the level of activity of the motor neuron in thepresence of the test compound with a level of activity of the motorneuron in the absence of the test compound; and selecting, identifying,purifying, or isolating the test compound as a candidate compound ifthere is a lower level of activity of the motor neuron in the presenceof the test compound than in its absence. In one embodiment, the methodscan include determining whether the candidate compound reduces a symptomof ALS in an animal model, e.g., a mouse model overexpressing humanmutant SOD1 G93A, wherein a candidate compound that reduces a symptom ofALS is a candidate compound to treat ALS.

In one embodiment, the motor neuron is obtained from or derived from asubject with a neurodegenerative disease, e.g., ALS, e.g., familial ALSor sporadic ALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, orpain. Determining a level of activity of the motor neuron can includemeasuring an action potential of the motor neuron, e.g., using patchclamp recording or extracellular multi-electrode array recording. In oneembodiment, the methods include determining whether the candidatecompound increases survival of the motor neuron.

In another aspect, methods of diagnosing a neurodegenerative disease,e.g., ALS, e.g., familial ALS or sporadic ALS, Parkinson's Disease,Alzheimer's Disease, epilepsy, or pain, are described. The methods caninclude providing a motor neuron from a subject; determining a level ofactivity of the motor neuron; comparing the level of activity of themotor neuron with a reference level of activity of a motor neuron; anddiagnosing the subject with a neurodegenerative disease if the level ofactivity of the motor neuron is higher than the reference level ofactivity.

In one embodiment, the motor neuron is derived from a subject with ALS,e.g., familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer'sDisease, epilepsy, or pain. In one embodiment, the methods can includemeasuring an action potential of the motor neuron, e.g., using patchclamp recording or extracellular multi-electrode array recording. In oneembodiment, the method can include administering to a subject diagnosedas having a neurodegenerative disease a therapeutically effective amountof a potassium channel opener, thereby treating the neurodegenerativedisease in the subject.

In yet another embodiment, methods of identifying a subject at increasedrisk of developing a neurodegenerative disease, e.g., ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, or pain, are provided. The methods can include providing amotor neuron from a subject; determining a level of activity of themotor neuron; comparing the level of activity of the motor neuron with areference level of activity of a motor neuron; and identifying thesubject at increased risk of developing a neurodegenerative disease ifthe level of activity of the motor neuron is higher than the referencelevel of activity. In one embodiment, the motor neuron is derived fromthe subject.

In one embodiment, the methods can include measuring an action potentialof the motor neuron, e.g., using patch clamp recording or extracellularmulti-electrode array recording. In one embodiment, the method caninclude administering to a subject identified as at increased risk ofdeveloping a neurodegenerative disease a therapeutically effectiveamount of a potassium channel opener, thereby reducing the risk ofdeveloping the neurodegenerative disease in the subject.

In one embodiment, the compound or pharmaceutical composition isadministered to the subject orally, intravenously, intrathecally,intraperitoneally, intramuscularly, or by implantation. In anotherembodiment, the methods further include administering to the subject ananti-neurodegenerative therapy, e.g., riluzole.

The present disclosure also features methods of treating, or reducing arisk of developing, a neurodegenerative disease, e.g., ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, or pain, in a subject. The methods include providing a somaticcell-derived motor neuron from a subject. In some embodiments, the motorneuron can be derived from a somatic cell, e.g., a fibroblast,lymphocyte, or keratinocyte, by reprogramming an induced pluripotentstem cell to differentiate into a functional motor neuron. In someembodiments, the somatic cell can be directly reprogrammed into afunctional motor neuron. The methods include determining a level ofactivity of the somatic cell-derived motor neuron and comparing thelevel of activity of the motor neuron with a reference level of activityof a motor neuron. The subject is identified as having, or at increasedrisk of developing, a neurodegenerative disease if the level of activityof the motor neuron is higher than the reference level of activity. Themethods include administering to the subject identified as having or atincreased risk of developing a neurodegenerative disease atherapeutically effective amount of a potassium channel opener, therebytreating or reducing the risk of developing the neurodegenerativedisease in the subject. In some embodiments, the potassium channelopener is a KCNQ/Kv7 channel opener, a K_(ATP) channel opener, a Gprotein-coupled inwardly-rectifying potassium channel opener, avoltage-gated Ca²⁺-activated potassium channel opener, or an inwardrectifier potassium channel opener. In some embodiments, the potassiumchannel opener is retigabine, e.g., a halogenated and/or a fluorinatedderivative of retigabine, meclofenamic acid, diclofenac, BMS-204352,diazoxide, minoxidil, nicorandil, pinacidil, levcromakalim, orflupirtine. In some embodiments, the methods also include administeringto the subject an anti-neurodegenerative therapy, e.g., riluzole.

In the methods described herein, the subject is an animal, human ornon-human, and rodent or non-rodent. For example, the subject can be anymammal, e.g., a human, other primate, pig, rodent such as mouse or rat,rabbit, guinea pig, hamster, cow, horse, cat, dog, sheep or goat, or anon-mammal such as a bird.

As used herein, treating or reducing a risk of developing aneurodegenerative disease in a subject means to ameliorate at least onesymptom of neurodegenerative disease. In one aspect, the inventionfeatures methods of treating, e.g., reducing severity or progression of,a neurodegenerative disease in a subject. The term treating can alsoinclude reducing the risk of developing a neurodegenerative disease in asubject, delaying the onset of symptoms of a neurodegenerative diseasein a subject, or increasing the longevity of a subject having aneurodegenerative disease. The methods can include selecting a subjecton the basis that they have, or are at risk of developing, aneurodegenerative disease, but do not yet have a neurodegenerativedisease, or a subject with an underlying neurodegenerative disease.Selection of a subject can include detecting symptoms of aneurodegenerative disease, a blood test, clinical electrophysiologicalrecordings, or imaging tests of the brain. If the results of the test(s)indicate that the subject has a neurodegenerative disease, the methodsalso include administering a therapeutically effective amount of apotassium channel opener, and detecting an effect of the potassiumchannel opener in the subject, thereby treating or reducing the risk ofdeveloping a neurodegenerative disease in the subject.

As used herein, the term “neurodegenerative disease” refers to acondition having a pathophysiological component of neuronal death.Neurodegeneration is the umbrella term for the progressive loss ofstructure or function of neurons, including death of neurons. Exemplaryexamples of such diseases include, but are not limited to, ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, and pain. ALS and its symptoms are well-known in the art andare described in further detail below. Subjects can be diagnosed ashaving a neurodegenerative disease by a health care provider, medicalcaregiver, physician, nurse, family member, or acquaintance, whorecognizes, appreciates, acknowledges, determines, concludes, opines, ordecides that the subject has a neurodegenerative disease.

The term “amyotrophic lateral sclerosis” or “ALS” also known as motorneuron disease and also as Lou Gehrig's disease, refers to a disease ofthe nerve cells in the brain and spinal cord that control voluntarymuscle movement. In ALS, neurons waste away or die, and can no longersend messages to muscles. This eventually leads to muscle weakening,twitching, and an inability to move the arms, legs, and body. Thecondition slowly gets worse. When the muscles in the chest area stopworking, it becomes hard or impossible to breathe on one's own. ALSaffects approximately five out of every 100,000 people worldwide. Asubject may be at risk for developing ALS if the subject has a familymember who has a hereditary form of the disease. Smoking and militaryexposure may be subtle risk factors. Symptoms usually do not developuntil after age 50, but they can start in younger people. Persons withALS have a loss of muscle strength and coordination that eventually getsworse and makes it impossible to do routine tasks such as going upsteps, getting out of a chair, or swallowing. Breathing or swallowingmuscles may be the first muscles affected. As the disease gets worse,more muscle groups develop problems. ALS does not affect the senses(sight, smell, taste, hearing, touch). It only rarely affects bladder orbowel function, or a person's ability to think or reason.

Parkinson's Disease occurs when nerve cells in the brain that makedopamine are slowly destroyed. Without dopamine, the nerve cells in thatpart of the brain cannot properly send messages. This leads to the lossof muscle function. The damage gets worse with time. The term“parkinsonism” refers to any condition that involves the types ofmovement changes seen in Parkinson's Disease. Parkinsonism may be causedby other disorders (called secondary parkinsonism) or certainmedications. Symptoms may be mild at first, and can include: slowblinking, constipation, difficulty swallowing, drooling, problems withbalance and walking, muscle aches and pains, rigid or stiff muscles, andshaking (tremors).

Alzheimer's Disease is a type of dementia that causes problems withmemory, thinking and behavior. Symptoms usually develop slowly and getworse over time, becoming severe enough to interfere with daily tasks.Alzheimer's Disease is the most common form of dementia, a general termfor memory loss and other intellectual abilities serious enough tointerfere with daily life. Alzheimer's Disease accounts for 50 to 80% ofdementia cases. Alzheimer's Disease is a progressive disease, wheredementia symptoms gradually worsen over a number of years. In its earlystages, memory loss is mild, but with late-stage Alzheimer's Disease,individuals lose the ability to carry on a conversation and respond totheir environment. Alzheimer's Disease is the sixth leading cause ofdeath in the United States. Those with Alzheimer's Disease live anaverage of eight years after their symptoms become noticeable to others,but survival can range from four to 20 years, depending on age and otherhealth conditions. Alzheimer's has no current cure, but treatments forsymptoms are available and research continues. Although currentAlzheimer's Disease treatments cannot stop Alzheimer's Disease fromprogressing, they can temporarily slow the worsening of dementiasymptoms and improve quality of life for those with Alzheimer's Diseaseand their caregivers. Today, there is a worldwide effort under way tofind better ways to treat the disease, delay its onset, and prevent itfrom developing.

Epilepsy is a common and diverse set of chronic neurological disorderscharacterized by seizures. Seizures may be recurrent and unprovoked,combined with brain alterations which increase the chance of futureseizures. In many cases a cause cannot be identified; however, braintrauma, strokes, brain cancer, and drug and alcohol misuse appear to beinvolved. Epileptic seizures result from abnormal, excessive, orhypersynchronous neuronal activity in the brain. About 50 million peopleworldwide have epilepsy, and nearly 80% of epilepsy occurs in developingcountries. Epilepsy becomes more common as people age. Onset of newcases occurs most frequently in infants and the elderly. Symptoms varyfrom person to person. Some people may have simple staring spells, whileothers have violent shaking and loss of alertness. The type of seizuredepends on the part of the brain affected and cause of epilepsy. Most ofthe time, the seizure is similar to the previous one. Some people withepilepsy have a strange sensation (such as tingling, smelling an odorthat isn't actually there, or emotional changes) before each seizure.Epilepsy is usually controlled, but not cured, with medication. However,more than 30% of people with epilepsy do not have seizure control evenwith the best available medications. Surgery may be considered indifficult cases.

By the phrase “risk of developing disease” is meant the relativeprobability that a subject will develop a neurodegenerative disease inthe future as compared to a control subject or population (e.g., ahealthy subject or population).

The term “potassium channel opener” is used to describe a type ofcompound that facilitates ion transmission through potassium channels.The methods include any potassium channel opener that acts on potassiumchannels expressed by normal or diseased neurons including motorneurons. These could be determined by mRNA expression profiling and/orelectrophysiological experiments. Examples include, but are not limitedto KCNQ/Kv7 channel openers, e.g., retigabine, e.g., a halogenatedand/or a fluorinated derivative of retigabine, meclofenamic acid,diclofenac, and BMS-204352; K_(ATP) channel openers, e.g., diazoxide,minoxidil, nicorandil, pinacidil, and levcromakalim; G protein-coupledinwardly-rectifying potassium channel openers, e.g., flupirtine; avoltage-gated Ca²⁺-activated potassium channel opener; or an inwardrectifier potassium channel opener.

The term “inhibitory RNA” is meant to include a nucleic acid moleculethat contains a sequence that is complementary to a target nucleic acid(e.g., a target microRNA or target inflammatory marker) that mediates adecrease in the level or activity of the target nucleic acid.Non-limiting examples of inhibitory RNAs include interfering RNA, shRNA,siRNA, ribozymes, antagomirs, and antisense oligonucleotides. Methods ofmaking inhibitory RNAs are described herein. Additional methods ofmaking inhibitory RNAs are known in the art.

As used herein, “an interfering RNA” refers to any double stranded orsingle stranded RNA sequence, capable—either directly or indirectly(i.e., upon conversion) of inhibiting or down-regulating gene expressionby mediating RNA interference. Interfering RNA includes, but is notlimited to, small interfering RNA (“siRNA”) and small hairpin RNA(“shRNA”). “RNA interference” refers to the selective degradation of asequence-compatible messenger RNA transcript.

As used herein “an shRNA” (small hairpin RNA) refers to an RNA moleculecomprising an antisense region, a loop portion and a sense region,wherein the sense region has complementary nucleotides that base pairwith the antisense region to form a duplex stem. Followingpost-transcriptional processing, the small hairpin RNA is converted intoa small interfering RNA by a cleavage event mediated by the enzymeDicer, which is a member of the RNase III family. As used herein, thephrase “post-transcriptional processing” refers to mRNA processing thatoccurs after transcription and is mediated, for example, by the enzymesDicer and/or Drosha.

A “small interfering RNA” or “siRNA” as used herein refers to any smallRNA molecule capable of inhibiting or down regulating gene expression bymediating RNA interference in a sequence specific manner. The small RNAcan be, for example, about 18 to 21 nucleotides long.

As used herein, an “antagomir” refers to a small synthetic RNA havingcomplementarity to a specific microRNA target, optionally with eithermispairing at the cleavage site or one or more base modifications toinhibit cleavage.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Methods and materials are described hereinfor use in the present invention; other, suitable methods and materialsknown in the art can also be used. The materials, methods, and examplesare illustrative only and not intended to be limiting. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. Otherfeatures and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a bar graph showing percentage of motor neurons able to fireaction potentials from control (18a and 11a) and ALS (39b and Rb9d)patient-derived motor neurons.

FIG. 2 shows action potentials recordings elicited by rampdepolarizations in control (18a and 11a) and ALS (39b and Rb9d)patient-derived motor neurons.

FIG. 3 is plot of spontaneous action potentials in extracellular MEArecording from two of 64 electrodes.

FIG. 4 is a graph of number of spikes in one minute of MEA recording.

FIG. 5 is a series of bar graphs depicting firing rate in one minute ofMEA recording.

FIG. 6 is a histogram of interspike interval from a single burstingALS-derived neuron in one minute of MEA recording

FIG. 7 shows delayed rectifier potassium current amplitudes (relative topeak sodium current) in control (18a and 11a) and ALS (39b and Rb9d)patient-derived motor neurons (p<0.05).

FIG. 8 is a Hill plot of block of spontaneous action potentials byretigabine in ALS patient-derived motor neurons (39b).

FIG. 9 depicts persistent sodium current plotted as a function ofvoltage during slow ramp depolarization in control (18a and 11a)compared to ALS (39b and Rb9d) patient-derived motor neurons (*p<0.01).

FIG. 10 is a bar graph showing that retigabine increased survival ofmotor neurons derived from ALS subjects.

FIG. 11 is a graph of number of spikes in one minute of MEA recording ofmotor neurons from four control patients (five lines), four SOD1patients, two C9orf72 repeat expansions, and two FUS patients.

FIG. 12 is a Hill plot of block of spontaneous action potentials byretigabine in C9orf72- and FUS-derived motor neurons.

DETAILED DESCRIPTION

The invention described herein is based in part on identification ofdecreased delayed rectifier potassium current and increased persistentsodium current in motor neurons derived from patients with aneurodegenerative disease, e.g., ALS, e.g., familial ALS or sporadicALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, or pain,compared to controls. The present methods can be used to treat or reducea risk of developing a neurodegenerative disease, including ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, or pain.

Methods of Treating or Reducing a Risk of Developing a NeurodegenerativeDisease

Provided herein are methods of treating or reducing a risk of developinga neurodegenerative disease (e.g., ALS, e.g., familial ALS or sporadicALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, or pain) thatinclude administering to a subject a therapeutically effective amount ofa potassium channel opener, e.g., retigabine, e.g., a halogenated and/ora fluorinated derivative of retigabine. The methods can involvediagnosing a subject, and administering, e.g., orally, to a subject,having or at risk for developing a neurodegenerative disease, atherapeutically effective amount of a potassium channel opener, e.g.,retigabine, e.g., a halogenated and/or a fluorinated derivative ofretigabine. The subject can be further monitored for treatment response.

Also featured in the present disclosure are methods of treating, orreducing a risk of developing, a neurodegenerative disease, e.g., ALS,e.g., familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer'sDisease, epilepsy, or pain, in a subject. The methods include providinga somatic cell-derived motor neuron from a subject. Motor neurons can bederived from, e.g., somatic cells (e.g., fibroblasts, lymphocytes,keratinocytes), from a subject and be reprogrammed to inducedpluripotent stem cells (iPSCs), which divide indefinitely in vitro andretain the ability to differentiate into any cell type (Dimos et al.,Science 321:1218-21, 2008; and Kiskinis et al., J Clin Invest 120:51-9,2010, which are hereby incorporated by reference in their entirety).These iPSCs can then be differentiated by an addition of small moleculesas described in Boulting et al. (Nat Biotechnol 29:279-86, 2011, whichis hereby incorporated by reference in its entirety) to functional motorneurons. Another way to derive motor neurons from a subject with aneurodegenerative disease is by direct reprogramming of somatic cells(e.g., fibroblasts) to functional motor neurons (Son et al., Cell StemCell 9:205-18, 2011, which is hereby incorporated by reference in itsentirety).

The methods include determining a level of activity of the somaticcell-derived motor neuron and comparing the level of activity of themotor neuron with a reference level of activity of a motor neuron. Thesubject is identified as having, or at increased risk of developing, aneurodegenerative disease if the level of activity of the motor neuronis higher than the reference level of activity. The methods also includeadministering to the subject identified as having or at increased riskof developing a neurodegenerative disease a therapeutically effectiveamount of a potassium channel opener, thereby treating or reducing therisk of developing the neurodegenerative disease in the subject. In someembodiments, the potassium channel opener is a KCNQ/Kv7 channel opener,a K_(ATP) channel opener, a G protein-coupled inwardly-rectifyingpotassium channel opener, a voltage-gated Ca²⁺-activated potassiumchannel opener, or an inward rectifier potassium channel opener. In someembodiments, the potassium channel opener is retigabine, e.g., ahalogenated and/or a fluorinated derivative of retigabine, meclofenamicacid, diclofenac, BMS-204352, diazoxide, minoxidil, nicorandil,pinacidil, levcromakalim, or flupirtine. In some embodiments, themethods also include administering to the subject ananti-neurodegenerative therapy, e.g., riluzole.

In some embodiments of any of the methods described herein, the subjectis suspected of having, is at risk of having, or has a neurodegenerativedisease, e.g., ALS, e.g., familial ALS or sporadic ALS, Parkinson'sDisease, Alzheimer's Disease, epilepsy, or pain. It is well within theskills of an ordinary practitioner to recognize a subject that has, oris at risk of developing, a neurodegenerative disease.

In all of the methods described herein, appropriate dosages of thepotassium channel opener can readily be determined by those of ordinaryskill in the art of medicine, e.g., by monitoring the patient for signsof disease amelioration or inhibition, and increasing or decreasing thedosage and/or frequency of treatment as desired. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue, e.g., motor neuron, in order to minimizepotential damage to unaffected cells and, thereby, reduce side effects.Appropriate doses can also be determined by amelioration of abnormalexcitability in peripheral nerves detected by clinical electrophysiologyor electromyography.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

For the compounds described herein, an effective amount (i.e., aneffective dosage) ranges from about 10 to 2000 mg/day, e.g., about 20 to1800 mg/day, e.g., about 30 to 1600 mg/day, e.g., about 50 to 1500mg/day, e.g., about 60 to 1200 mg/day, e.g., about 100 to 1000 mg/day,e.g., about 200 mg/day. Optimal dosage levels can be readily determinedby a skilled practitioner, such as a physician, e.g., a neurologist. Thecompound can be administered one time per day, twice per day, one timeper week, twice per week, for between about 1 to 52 weeks per year,e.g., between 2 to 50 weeks, about 6 to 40 weeks, or for about 4, 5, or6 weeks. The skilled artisan will appreciate that certain factorsinfluence the dosage and timing required to effectively treat a patient,including but not limited to the type of patient to be treated, theseverity of the disease or disorder, previous treatments, the generalhealth and/or age of the patient, and other diseases present. Moreover,treatment of a patient with a therapeutically effective amount of acompound can include a single treatment or, preferably, can include aseries of treatments.

For example, for retigabine, e.g., a halogenated and/or a fluorinatedderivative of retigabine, an effective amount ranges from about 100mg/day 200 mg/day, about 400 mg/day, about 500 mg/day, about 600 mg/day,about 800 mg/day, about 900 mg/day, about 1000 mg/day, and about 2000mg/day.

A subject can be treated (e.g., periodically administered the agent) fora prolonged period of time (e.g., at least one month, two months, sixmonths, one year, two years, three years, four years, five years, or tenyears). As described in detail herein, the dosage of the potassiumchannel opener to be administered to the subject can be determined by aphysician by consideration of a number of physiological factorsincluding, but not limited to, the sex of the subject, the weight of thesubject, the age of the subject, and the presence of other medicalconditions. The potassium channel opener can be administered to thesubject orally, intravenously, intrathecally, intraperitoneally,intramuscularly, or by implantation with appropriate change in dosage toreach desired EC50 levels.

The subjects can also be those undergoing any of a variety of additionalanti-neurodegenerative therapy treatments. Thus, for example, subjectscan be those being treated with riluzole.

Neurodegenerative Diseases

Neurodegenerative diseases are a class of neurological diseases that arecharacterized by the progressive loss of the structure and function ofneurons and neuronal cell death. Inflammation has been implicated for arole in several neurodegenerative diseases. Progressive loss of motorand sensory neurons and the ability of the mind to refer sensoryinformation to an external object is affected in different kinds ofneurodegenerative diseases. Non-limiting examples of neurodegenerativediseases include ALS, e.g., familial ALS or sporadic ALS, Parkinson'sDisease, Alzheimer's Disease, epilepsy, or pain.

A health care professional may diagnose a subject as having aneurodegenerative disease by the assessment of one or more symptoms of aneurodegenerative disease in the subject. Non-limiting symptoms of aneurodegenerative disease in a subject include difficulty lifting thefront part of the foot and toes; weakness in arms, legs, feet, orankles; hand weakness or clumsiness; slurring of speech; difficultyswallowing; muscle cramps; twitching in arms, shoulders, and tongue;difficulty chewing; difficulty breathing; muscle paralysis; partial orcomplete loss of vision; double vision; tingling or pain in parts ofbody; electric shock sensations that occur with head movements; tremor;unsteady gait; fatigue; dizziness; loss of memory; disorientation;misinterpretation of spatial relationships; difficulty reading orwriting; difficulty concentrating and thinking; difficulty makingjudgments and decisions; difficulty planning and performing familiartasks; depression; anxiety; social withdrawal; mood swings;irritability; aggressiveness; changes in sleeping habits; wandering;dementia; loss of automatic movements; impaired posture and balance;rigid muscles; bradykinesia; slow or abnormal eye movements; involuntaryjerking or writhing movements (chorea); involuntary, sustainedcontracture of muscles (dystonia); lack of flexibility; lack of impulsecontrol; and changes in appetite. A health care professional may alsobase a diagnosis, in part, on the subject's family history of aneurodegenerative disease. A health care professional may diagnose asubject as having a neurodegenerative disease upon presentation of asubject to a health care facility (e.g., a clinic or a hospital). Insome instances, a health care professional may diagnose a subject ashaving a neurodegenerative disease while the subject is admitted in anassisted care facility. Typically, a physician diagnoses aneurodegenerative disease in a subject after the presentation of one ormore symptoms.

Methods for Identifying Compounds to Treat Neurodegenerative Diseases

The present disclosure also provides methods for identifying compounds,e.g., small organic or inorganic molecules (e.g., molecules having amolecular weight less than 1,000 Da), oligopeptides, oligonucleotides,or carbohydrates, capable of reducing motor neuron activity and,therefore, treating neurodegenerative diseases.

Libraries of Test Compounds

In certain embodiments, screens of the present invention utilizelibraries of test compounds. As used herein, a “test compound” can beany chemical compound, for example, a macromolecule (e.g., apolypeptide, a protein complex, glycoprotein, or a nucleic acid) or asmall molecule (e.g., an amino acid, a nucleotide, an organic orinorganic compound). A test compound can have a formula weight of lessthan about 10,000 grams per mole, less than 5,000 grams per mole, lessthan 1,000 grams per mole, or less than about 500 grams per mole. Thetest compound can be naturally occurring (e.g., an herb or a naturalproduct), synthetic, or can include both natural and syntheticcomponents. Examples of test compounds include peptides, peptidomimetics(e.g., peptoids), amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, inhibitoryRNAs, shRNAs, small interfering RNAs, antagomirs, and organic orinorganic compounds, e.g., heteroorganic or organometallic compounds.

Test compounds can be screened individually or in parallel. An exampleof parallel screening is a high throughput drug screen of largelibraries of chemicals. Such libraries of candidate compounds can begenerated or purchased, e.g., from Chembridge Corp., San Diego, Calif.Libraries can be designed to cover a diverse range of compounds. Forexample, a library can include 500, 1000, 10,000, 50,000, or 100,000 ormore unique compounds. Alternatively, prior experimentation andanecdotal evidence can suggest a class or category of compounds ofenhanced potential. A library can be designed and synthesized to coversuch a class of chemicals.

The synthesis of combinatorial libraries is well known in the art andhas been reviewed (see, e.g., E. M. Gordon et al., J. Med. Chem. (1994)37:1385-1401; DeWitt, S. H.; Czarnik, A. W. Acc. Chem. Res. (1996)29:114; Armstrong, R. W.; Combs, A. P.; Tempest, P. A.; Brown, S. D.;Keating, T. A. Acc. Chem. Res. (1996) 29:123; Ellman, J. A. Acc. Chem.Res. (1996) 29:132; Gordon, E. M.; Gallop, M. A.; Patel, D. V. Acc.Chem. Res. (1996) 29:144; Lowe, G. Chem. Soc. Rev. (1995) 309, Blondelleet al. Trends Anal. Chem. (1995) 14:83; Chen et al. J. Am. Chem. Soc.(1994) 116:2661; U.S. Pat. Nos. 5,359,115, 5,362,899, and 5,288,514; PCTPublication Nos. WO92/10092, WO93/09668, WO91/07087, WO93/20242, andWO94/08051).

Libraries of compounds can be prepared according to a variety ofmethods, some of which are known in the art. For example, a “split-pool”strategy can be implemented in the following way: beads of afunctionalized polymeric support are placed in a plurality of reactionvessels; a variety of polymeric supports suitable for solid-phasepeptide synthesis are known, and some are commercially available (forexamples, see, e.g., M. Bodansky “Principles of Peptide Synthesis,” 2ndedition, Springer-Verlag, Berlin (1993)). To each aliquot of beads isadded a solution of a different activated amino acid, and the reactionsare allowed to proceed to yield a plurality of immobilized amino acids,one in each reaction vessel. The aliquots of derivatized beads are thenwashed, “pooled” (i.e., recombined), and the pool of beads is againdivided, with each aliquot being placed in a separate reaction vessel.Another activated amino acid is then added to each aliquot of beads. Thecycle of synthesis is repeated until a desired peptide length isobtained. The amino acid residues added at each synthesis cycle can berandomly selected; alternatively, amino acids can be selected to providea “biased” library, e.g., a library in which certain portions of theinhibitor are selected non-randomly, e.g., to provide an inhibitorhaving known structural similarity or homology to a known peptidecapable of interacting with an antibody, e.g., the an anti-idiotypicantibody antigen binding site. It will be appreciated that a widevariety of peptidic, peptidomimetic, or non-peptidic compounds can bereadily generated in this way.

The “split-pool” strategy can result in a library of peptides, e.g.,modulators, which can be used to prepare a library of test compounds ofthe invention. In another illustrative synthesis, a “diversomer library”is created by the method of Hobbs DeWitt et al. (Proc. Natl. Acad. Sci.USA 90:6909 (1993)). Other synthesis methods, including the “tea-bag”technique of Houghten (see, e.g., Houghten et al., Nature 354:84-86,1991) can also be used to synthesize libraries of compounds according tothe subject invention.

Libraries of compounds can be screened to determine whether any membersof the library have a desired activity, and, if so, to identify theactive species. Methods of screening combinatorial libraries have beendescribed (see, e.g., Gordon et al., J Med. Chem., supra). Compoundlibraries can be screened by providing a motor neuron from a subjectwith a neurodegenerative disease, e.g., a motor neuron derived from asubject with ALS, e.g., familial ALS or sporadic ALS, Parkinson'sDisease, Alzheimer's Disease, epilepsy, or pain, contacting the motorneuron with a test compound, determining a level of activity of themotor neuron, and comparing the level of activity of the motor neuron inthe presence of the test compound with a level of activity of the motorneuron in the absence of the test compound, wherein a lower level ofactivity of the motor neuron in the presence of the test compound thanin its absence indicates that the test compound is a candidate compoundto treat ALS, e.g., familial ALS or sporadic ALS, Parkinson's Disease,Alzheimer's Disease, epilepsy, or pain. The level of activity of themotor neuron can be determined by measuring an action potential of themotor neuron, e.g., using patch clamp recording or extracellularmulti-electrode array (MEA) recording.

Exemplary assays useful for screening libraries of test compounds aredescribed herein.

Screening Methods

The invention provides methods for identifying compounds capable ofmodulating motor neuron activity. Although applicants do not intend tobe bound by any particular theory as to the biological mechanisminvolved, such compounds are thought to specifically (1) increasedelayed rectifier potassium current and/or (2) decrease persistentsodium current in motor neurons.

In certain aspects of the present invention, screening for suchcompounds is accomplished by (i) contacting a motor neuron from asubject with ALS, e.g., a motor neuron derived from a subject with ALSwith a test compound; determining a level of activity of the motorneuron; and comparing the level of activity of the motor neuron in thepresence of the test compound with a level of activity of the motorneuron in the absence of the test compound. Test compounds that lowerthe level of activity of the motor neuron are referred to herein as“candidate compounds.” Candidate compounds can be further tested andfound to be capable of reducing in vivo activity of a motor neuron.

As described herein, motor neurons can be derived from a subject with aneurodegenerative disease, e.g., ALS, e.g., familial ALS or sporadicALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, or pain, byart-known methods. For example, somatic cells (e.g., fibroblasts,lymphocytes, keratinocytes) from a subject with ALS can be reprogrammedto induced pluripotent stem cells (iPSCs), which divide indefinitely invitro and retain the ability to differentiate into any cell type (Dimoset al., Science 321:1218-21, 2008; Kiskinis et al., J Clin Invest120:51-9, 2010). These iPSCs can then be differentiated by an additionof small molecules as described in Boulting et al. (Nat Biotechnol29:279-86, 2011) to functional motor neurons. Another way to derivemotor neurons from a subject with ALS is by direct reprogramming ofsomatic cells (e.g., fibroblasts) to functional motor neurons (Son etal., Cell Stem Cell 9:205-18, 2011).

Motor neuron activity could be assayed in a high-throughput system,recording large numbers (hundreds) of neurons within each well of amulti-well MEA device (such as from Axion systems which can use a 96well format) or recording individual neurons from a low throughputtechnique such as patch clamp. As an example of high-throughput usage,motor neuron activity can be recorded in 96 wells pre-filled withindividual compounds, different doses of compounds, or a combination ofcompounds. Assessment of activity may also be made by calcium imaging,which can be used in both low- and high-throughput formats.

In vivo testing of candidate compounds can be performed by means knownto those in the art. For example, the candidate compound(s) can beadministered to a mammal, such as a rodent (e.g., murine) or rabbit.Such animal model systems are art-accepted for testing potentialpharmaceutical agents to determine their therapeutic efficacy inpatients, e.g., human patients. Animals that are particularly useful forin vivo testing are wild type animals or non-wild type animals (e.g.,mice) that over-produce mutant human SOD1 (SOD1^(G93A)). In a typical invivo assay, an animal (e.g., a wild type or transgenic mouse) isadministered, by any route deemed appropriate (e.g., by injection), adose of a candidate compound. Conventional methods and criteria can thenbe used to monitor animals for signs of reduction of motor neuronactivity. If needed, the results obtained in the presence of thecandidate compound can be compared with results in control animals thatare not treated with the test compound.

The level of activity of a motor neuron can be determined by measuringan action potential of the motor neuron. Action potentials can bemeasured by patch clamp recording or extracellular MEA recording. Patchclamp recording is a laboratory technique that allows the study ofsingle or multiple ion channels in cells. The technique can be appliedto a wide variety of cells, but is especially useful in the study ofexcitable cells such as neurons. Patch clamp recording makes it possibleto record the currents of single ion channels.

Patch clamp recording uses, as an electrode, a glass micropipette thathas an open tip diameter of about one micrometer. The interior of themicropipette is filled with a solution matching the ionic composition ofthe bath solution, as in the case of cell-attached recording, or thecytoplasm for whole-cell recording. A chlorided silver wire is placed incontact with this solution and conducts electric current to theamplifier. The micropipette is pressed against a cell membrane andsuction is applied to assist in the formation of a high resistance sealbetween the glass and the cell membrane. The high resistance of thisseal makes it possible to electronically isolate the currents measuredacross the membrane patch with little competing noise, as well asproviding some mechanical stability to the recording.

Patch clamp recording uses a single electrode to record currents. Manypatch clamp amplifiers do not use true voltage clamp circuitry butinstead are differential amplifiers that use the bath electrode to setthe zero current level. This allows a researcher to keep the voltageconstant while observing changes in current. Alternatively, the cell canbe current clamped in whole-cell mode, keeping current constant whileobserving changes in membrane voltage.

Extracellular MEAs are devices that contain multiple plates or shanksthrough which neural signals are obtained or delivered, essentiallyserving as neural interfaces that connect neurons to electroniccircuitry. When recording, the electrodes on an MEA transduce a changein voltage from the environment carried by ions into currents carried byelectrons. The size and shape of a recorded signal depend upon severalfactors: the nature of the medium in which the cell or cells are located(e.g., the medium's electrical conductivity, capacitance, andhomogeneity); the nature of contact between the cells and the MEAelectrode (e.g., area of contact and tightness); the nature of the MEAelectrode itself (e.g., its geometry, impedance, and noise); the analogsignal processing (e.g., the system's gain, bandwidth, and behavioroutside of cutoff frequencies); and the data sampling properties (e.g.,sampling rate and digital signal processing). For the recording of asingle cell that partially covers a planar electrode, the voltage at thecontact pad is approximately equal to the voltage of the overlappingregion of the cell and electrode multiplied by the ratio the surfacearea of the overlapping region to the area of the entire electrode.

Drugs detected from their effects on excitability on human motor neuronsfrom subjects with a neurodegenerative disease, e.g., ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, or pain, can be tested for their effects on motor neuronsurvival in vitro.

Medicinal Chemistry

Once a compound (or agent) of interest has been identified, standardprinciples of medicinal chemistry can be used to produce derivatives ofthe compound. Derivatives can be screened for improved pharmacologicalproperties, for example, efficacy, pharmaco-kinetics, stability,solubility, and clearance. The moieties responsible for a compound'sactivity in the assays described above can be delineated by examinationof structure-activity relationships (SAR) as is commonly practiced inthe art. A person of ordinary skill in pharmaceutical chemistry couldmodify moieties on a candidate compound or agent and measure the effectsof the modification on the efficacy of the compound or agent to therebyproduce derivatives with increased potency. For an example, seeNagarajan et al. (1988) J. Antibiot. 41: 1430-8. Furthermore, if thebiochemical target of the compound (or agent) is known or determined,the structure of the target and the compound can inform the design andoptimization of derivatives. Molecular modeling software is commerciallyavailable (e.g., from Molecular Simulations, Inc.) for this purpose.

Diagnostic Methods

Also provided herein are methods of diagnosing a neurodegenerativedisease, e.g., ALS, e.g., familial ALS or sporadic ALS, Parkinson'sDisease, Alzheimer's Disease, epilepsy, or pain, in a subject byproviding a motor neuron from a subject with a neurodegenerativedisease, a subject suspected of having a neurodegenerative disease, or asubject at risk for a neurodegenerative disease, and determining a levelof activity of the motor neuron. The level of activity of the motorneuron can be compared to a reference level of activity of a motorneuron, e.g., motor neuron activity from a healthy cohort. A subjectwith a motor neuron with a higher level of activity than a referencelevel of activity indicates that the subject has a neurodegenerativedisease as outlined in detail below.

In some embodiments, a subject can be diagnosed as having ALS, e.g.,familial ALS or sporadic ALS, Parkinson's Disease, Alzheimer's Disease,epilepsy, or pain, if the level of activity of a motor neuron from thesubject is higher than a reference level of activity. In someembodiments, a subject diagnosed as having a neurodegenerative disease,e.g., ALS, e.g., familial ALS or sporadic ALS, Parkinson's Disease,Alzheimer's Disease, epilepsy, or pain, can be administered atherapeutically effective amount of a potassium channel opener, e.g.,KCNQ/Kv7 channel openers, e.g., retigabine, e.g., a halogenated and/or afluorinated derivative of retigabine, meclofenamic acid, diclofenac, andBMS-204352; K_(ATP) channel openers, e.g., diazoxide, minoxidil,nicorandil, pinacidil, and levcromakalim; G protein-coupledinwardly-rectifying potassium channel openers, e.g., flupirtine;voltage-gated Ca²⁺-activated potassium channel openers; and/or inwardrectifier potassium channel openers, thereby treating or reducing therisk of developing the neurodegenerative disease in the subject.

Any of the methods described herein can be performed on subjectspresenting to a health care facility (e.g., a hospital, clinic, or anassisted care facility). The subjects may present with one or moresymptoms of a neurodegenerative disease (e.g., any of the symptoms of aneurodegenerative disease described herein). The subject can alsopresent with no symptoms (an asymptomatic subject) or just one symptomof a neurodegenerative disease. The subject can have a familial historyof a neurodegenerative disease (e.g., familial ALS).

The diagnostic methods described herein can be performed by any healthcare professional (e.g., a physician, a laboratory technician, a nurse,a physician's assistant, and a nurse's assistant). The diagnosticmethods described herein can be used in combination with any additionaldiagnostic testing methods known in the art (e.g., the observation orassessment of one or more symptoms of a neurodegenerative disease in asubject).

Methods of Identifying a Subject at Risk of Developing aNeurodegenerative Disease

Also provided are methods of identifying a subject at risk of developinga neurodegenerative disease, e.g., ALS, e.g., familial ALS or sporadicALS, Parkinson's Disease, Alzheimer's Disease, epilepsy, or pain. Thesemethods include providing a motor neuron from a subject and determininga level of activity of the motor neuron. The level of activity of themotor neuron can be compared to a reference level of activity of a motorneuron, e.g., motor neuron activity from a healthy cohort. A subjectwith a motor neuron with a higher level of activity than a referencelevel of activity indicates that the subject is at risk of developing aneurodegenerative disease.

The subjects may present with one or more symptoms of aneurodegenerative disease (e.g., any of the symptoms of aneurodegenerative disease described herein). The subject can alsopresent with no symptoms or just one symptom of a neurodegenerativedisease. The subject can have a family history of a neurodegenerativedisease (e.g., familial ALS).

Subjects identified as at risk of developing a neurodegenerative diseasemay be administered a treatment for a neurodegenerative disease or maybe administered a new or alternative treatment for a neurodegenerativedisease. Subjects identified as at risk of developing aneurodegenerative disease can also undergo more aggressive therapeutictreatment (e.g., increased periodicity of clinic or hospital visits).

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims. Theexamples involve ALS, although skilled practitioners will appreciatethat methods may be applied to other neurodegenerative diseases, e.g.,Parkinson's Disease, Alzheimer's Disease, epilepsy, and pain.

Example 1 Action Potential Firing Properties of Motor Neurons Derivedfrom Patients with ALS and Age-Matched Control Subjects

Experiments were performed using iPS-derived motor neurons from twofamilial ALS subjects, both of whom harbored the same extremelyaggressive SOD1^(A4V) mutation (39b, Rb9d, both with disease onset inthe 40s and death within one-two additional years), and two separateage-matched control subjects (11a, 18a). All lines were determined to bekaryotypically normal, and all were efficient in motor neurondifferentiation (˜50%). Using two separate techniques, patch clamp andextracellular MEA recording, an increased excitability of motor neuronsderived from familial ALS patients compared to controls was found (FIG.1). During ramp depolarizations in current clamp, the number of actionpotentials fired by ALS motor neurons was larger than in control motorneurons (p<0.05, Mann-Whitney U test) (FIG. 2). Input resistance,capacitance, and resting membrane potential were not different among thegroups, suggesting that these findings were not related to, for example,poor health of the familial ALS-derived motor neurons. These resultswere obtained in experiments from three separate differentiations,arguing against a result of a poor differentiation of a particular line.

Because patch clamp experiments can only evaluate relatively smallnumbers of cells, extracellular MEA recording was used to evaluatespontaneous firing in large numbers of neurons, and a robust increase inthe number of spontaneous action potentials in familial ALS-derivedmotor neurons was found (FIGS. 3-4, p<0.05 Mann-Whitney U test, andexperimental details below). A 2-3 fold increase in the number ofspontaneously firing neurons in the familial ALS-derived neuronscompared to control-derived neurons was observed; moreover, a largernumber of mutant neurons than controls fired at fast frequencies (FIG.5), thus accounting for the large difference in spike number. Similarresults have been obtained in two to three separate motor neurondifferentiations.

Motor neurons from two aggressive familial ALS lines and two controlshave been evaluated. Patch clamp will be used to evaluate motor neuronsfrom two additional aggressive SOD1 familial ALS cases (two early onsetG85S patients, four total), four sporadic ALS cases (screened for knownfamilial ALS mutations, four total), and two additional controls (fourtotal). A large number of karyotypically normal iPSC lines from sporadicALS, familial ALS, and controls (nearly 100 disease and controlsubjects, and growing) are available, as well as extensive experience indifferentiating the lines into motor neurons (Boulting et al., NatBiotechnol 2011: 29, 279-86). Accompanying each ALS line is informationregarding patient age of onset, an indicator of disease severity.Differentiation protocol is based on dual-SMAD inhibition (Chambers etal., Nat Biotechnol 2009: 27, 275-80) and modification of the originalmorphogen-based approach (Wichterle et al., Cell 2002: 110, 385-97). Anequal number of disease and control lines will be differentiated intomotor neurons at each time in order to control as much as possible forvariation in differentiation efficiency. Data from each cell line willbe pooled from at least three separate differentiations. All recordingswill take place at approximately four weeks in culture, as at this timepoint well-developed motor neuron electrical activity and substantialnumbers of surviving motor neurons have been observed in culture.

Extracellular recordings will be performed using a MED64 MEA amplifierto record from an 8×8 array of extracellular electrodes (Alpha MedScientific) after plating equal numbers of cells on each array. Becauseeach action potential has a distinct morphology, spike-clusteringsoftware can be used to determine the number of spontaneously firingneurons (Lewicki, Network 1998: 9, R53-78; Cohen et al., Nat Neurosci2011: 14, 811-9). Custom Matlab programs have been written to ensurethat neurons whose spikes are detected by multiple electrodes are onlycounted once. Average spiking frequency for each neuron and individualinterspike intervals (ISI) between consecutive spikes of each neuronwill be calculated. A non-bursting neuron should have a distribution ofISIs described by a single Poisson distribution, while a bursting neuronwill have a bimodal distribution of ISIs (or log of ISI), as shown inFIG. 6. Individual bursting and non-bursting neurons will be identifiedbased on deviation from Poisson fits of ISIs for particular neurons(Bastian et al., Journal of Neurophysiology 2001: 85, 10-22) and thenumber and percentage of bursting neurons will be quantified.

Example 2 Phenotype the Major Classes of Ion Channels and ReceptorsPresent in iPSC-Derived Motor Neurons and Determine Whether ReducingChannel Activity Improves Motor Neuron Survival In Vitro

In repeated differentiations of motor neurons from the two control andtwo SOD1^(A4V) iPSC lines, the relative amplitude of the steady-statedelayed rectifier potassium current was markedly smaller in diseasecompared to control motor neurons. The relative amplitudes werequantified, and a significant decrease was found between control andfamilial ALS-derived motor neurons (FIG. 7, p<0.05, t-test).

Retigabine, an FDA-approved activator of Kv7 currents (although athigher concentrations, it may potentiate GABA responses), suppressedspontaneous firing as assessed by MEA recording at 100 nM, aconcentration thought to be physiologically relevant to itspharmacological activity in patients and similar to its documented EC50on Kv7 channels (Ferron et al., Br J Clin Pharmacol 2003: 56, 39-45;Wickenden et al., Mol Pharmacol 2000: 58, 591-600) (FIG. 8). While theHill plot was from a single MEA (˜300 active neurons), 100% were blockedwith 10 μM on two additional MEAs from separate differentiations. Inaddition, RT-PCR data from the neurons show high Kv7 mRNA levels.

Small persistent sodium currents that were larger in ALS-derived motorneurons than controls have been measured (FIG. 9, p<0.05, t-test). Thisfinding was not due to differences in cell size, as the capacitancemeasurements were not different between the groups. While these currentsare small, the fact that the persistent sodium current does notinactivate means that it still can potentially have a potent effect onneuronal firing.

Experiments have been performed in which no difference in amplitudes ofresponses to the excitatory neurotransmitter kainate or to theinhibitory neurotransmitters GABA and glycine have been observed.

Example 3 Test Retigabine and Other Test Compounds in the SOD1^(G93A)Mouse Model

Retigabine and other test compounds will be tested to determine whetherthey delay disease onset or reduce mortality in SOD1^(G93A) mice. Othertests will also be used to detect improved motor function, force,reduced motor neuron or ventral root loss, maintained innervation, orpreserved muscle mass, all of which are affected in SOD1^(G93A) mice.Gurney et al., Science 1994: 264, 1772-5; Fischer et al., Exp Neurol2004: 185, 232-40; and Mead et al., PLoS ONE 2011: 6, e23244). The onsetof disease in SOD1G93A mice is classically reported around 90 days ofage, and death usually occurs at 130-140 days. Crude signs of diseaseonset typically involve tremor in one limb. However, rotarod performancehas been found to decline several weeks before such manifestations.

Because the mice have been used extensively in drug studies, there areconsensus guidelines for conducting studies and reliable estimates ofthe numbers needed to obtain statistically meaningful results (Ludolphet al., Amyotroph Lateral Scler 2010: 11, 38-45; and Mead et al., PLoSONE 2011: 6, e23244). 15 animals per group will allow detection of a 20%improvement in rotarod performance, a value thought to be biologicallyrelevant and a sensitive indicator of disease onset, with 80% power at asignificance level of 0.05. Additional outcomes require 5-7 animals pergroup to obtain biologically relevant improvements with 80% power andsignificance level 0.05. 60 four week-old high copy number SOD1^(G93A)transgenic mice (Jackson 002726) will be divided randomly into twogroups, one to receive active drug and the second to receive vehicle.Each group of 30 mice will randomly be divided into two again, 15animals for behavior/survival and 15 for histology, with full blindingfor all experiments and analysis. Equal numbers of males and femaleswill be maintained for all comparisons. Transgene copy numbers will bechecked only if reduction is suspected by phenotype (Mead et al., PLoSONE 2011: 6, e23244).

Retigabine has recently been used in mice treated with doses from 1-10mg/kg given via intraperitoneal (IP) injection, and studies of the drugin epilepsy models have documented CSF uptake (mass spectrometry ofspinal cord could be used if necessary). In a study evaluatingretigabine to treat peripheral neuropathy, mice were treated with 10mg/kg IP injection for several weeks without apparent side effects(Nodera et al., Neuroscience Letters 2011: 505, 223-7). We will treatmice with IP injection of vehicle or 10 mg/kg retigabine five days perweek from age four weeks until death.

Analyses for histological and behavioral tests are performed usingt-tests between groups with appropriate correction for multiplecomparisons. Disease onset and survival curves are analyzed by KaplanMeir and Logrank Mantel-Cox tests, respectively. Five mice in each groupare sacrificed for histological experiments at each time point of 60,90, and 120 days (30 mice total). Motor neuron quantification,neuromuscular junction (NMJ) analysis, ventral root counts, and muscleweights are performed with these five mice/group. For motor neuronquantification, Image J software is used to detect NeuN-stained lumbarventral-horn neurons with area>450 μm², a standard criteria for motorneurons. For NMJ analysis, the gastrocnemius muscles are excised,weighed, and co-stained sections with antibodies to neurofilament 200and labeled alpha-bungarotoxin. NMJs are identified based onbungarotoxin staining and pretzel-shaped morphology, and the number ofNMJs are counted, as well as the number of innervated NMJs based onoverlap of bungarotoxin and neurofilament signals. The number of NMJs,percent innervated and percent denervated are quantified. For ventralroot counts, thin sections are stained with toluidine blue, large andsmall fiber axons counted with Image J software, and the absolute numberand percentage of small and large fiber axons are quantified. The second30 mice are used for behavioral and survival studies (n=15 in eachgroup). Mice behavior is analyzed starting at 40 days of age and testsperformed biweekly. Animal weight is recorded. Maximum muscle force ismeasured musing grip strength analysis and motor function using anaccelerating rotarod. These behavioral tests are commonly used and willbe performed in triplicate.

Effects of retigabine on wild-type littermates in both behavior andhistology will be controlled for using five animals per group forbehavior and five for histology analyzed at 120 days only (10 treated,10 untreated).

Example 4 Retigabine Increased Survival of Motor Neurons Derived fromALS Subjects

ALS patient-derived motor neurons were identified by positive stain forISL and TUJ1 after 30 days in culture with or without retigabine (10μM). The drug increased the survival of the motor neurons (FIG. 10).

Example 5 Retigabine Increased Survival of Motor Neurons Derived fromALS Subjects

SOD1-derived motor neurons (four lines from four individual unrelatedsubjects), C9orf72-derived motor neurons (two lines from two individualunrelated subjects), FUS-derived motor neurons (four lines from twoindividual unrelated subjects) were hyperexcitable compared to motorneurons derived from seven iPSC lines made from five individual healthycontrols (FIG. 11). Retigabine blocks the firing of the C9orf72 andFUS-derived motor neurons (FIG. 12). iPSC line and motor neurongeneration for these experiments were performed exactly as for thepreviously studied SOD1^(A4V) and control lines. All lines werekaryotypically normal, and all were efficient in motor neurondifferentiation. 25b contains a SOD1^(D90A) mutation; 27d contains aSOD1^(G85S) mutation; MGH5b contains a frameshift mutation at FUSresidue 1529; and RB21 contains a H517Q FUS mutation. Both 19f and RB8BiPSC lines were generated from patient fibroblasts that carry anextended number of repeat expansions in the gene C9orf72. All iPSCs aregenerated via 3-factor (OCT4/SOX2/KLF4) retroviral reprogramming aspreviously described in Dimos et al. (Science 321:1218-21, 2008); andBoulting et al. (Nat Biotechnol 29:279-86, 2011). Line RB8B wasgenerated by 4-factor reprogramming (OCT4/SOX2/KLF4/cMYC) in the samemanner. All lines have been quality controlled for pluripotency usingstandard assays, including staining for NANOG and the Scorecard analysisas described in Bock et al. (Cell 144:439-452, 2011). Control lines 11a,15b, 17a, 18a, 18b and 20b have previously been published in Boulting etal. (Nat Biotechnol 29:279-86, 2011).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of diagnosing a subject with amyotrophiclateral sclerosis (ALS), the method comprising: providing a motor neuronfrom the subject; determining a level of activity of the motor neuron;comparing the level of activity of the motor neuron with a referencelevel of activity of a motor neuron; and diagnosing the subject ashaving ALS if the level of activity of the motor neuron is higher thanthe reference level of activity.
 2. The method of claim 1, whereindetermining a level of activity of the motor neuron comprises measuringan action potential of the motor neuron.
 3. The method of claim 1,wherein determining a level of activity of the motor neuron comprisespatch clamp recording or extracellular multi-electrode array recording.4. A method of treating, or reducing a risk of developing, ALS in asubject, the method comprising: providing a motor neuron from a subject;determining a level of activity of the motor neuron; comparing the levelof activity of the motor neuron with a reference level of activity of amotor neuron; identifying the subject as having, or at increased risk ofdeveloping, ALS if the level of activity of the motor neuron is higherthan the reference level of activity; and administering to the subjectidentified as having or at increased risk of developing ALS atherapeutically effective amount of a potassium channel opener, therebytreating or reducing the risk of developing ALS in the subject.
 5. Themethod of claim 4, wherein the ALS is familial ALS or sporadic ALS. 6.The method of claim 4, wherein the potassium channel opener is aKCNQ/Kv7 channel opener, a KATP channel opener, a G protein-coupledinwardly-rectifying potassium channel opener, a voltage-gatedCa2+-activated potassium channel opener, or an inward rectifierpotassium channel opener.
 7. The method of claim 4, wherein thepotassium channel opener is retigabine, a halogenated derivative ofretigabine, a fluorinated derivative of retigabine, meclofenamic acid,diclofenac, BMS-204352, diazoxide, minoxidil, nicorandil, pinacidil,levcromakalim, or flupirtine.
 8. The method of claim 4, wherein thepotassium channel opener is retigabine.
 9. The method of claim 4, themethod further comprising administering to the subject ananti-neurodegenerative therapy.
 10. The method of claim 9, wherein theanti-neurodegenerative therapy is riluzole.
 11. A method of identifyinga candidate compound to treat ALS, the method comprising: providing amotor neuron from a subject with ALS or a motor neuron derived from asubject with ALS; contacting the motor neuron with a test compound;determining a level of activity of the motor neuron; comparing the levelof activity of the motor neuron in the presence of the test compoundwith a level of activity of the motor neuron in the absence of the testcompound; and selecting the test compound as a candidate compound ifthere is a lower level of activity of the motor neuron in the presenceof the test compound than in its absence.
 12. The method of claim 11,wherein determining a level of activity of the motor neuron comprisesmeasuring an action potential of the motor neuron.
 13. The method ofclaim 11, wherein determining a level of activity of the motor neuroncomprises patch clamp recording or extracellular multi-electrode arrayrecording.
 14. The method of claim 11, wherein the test compound isselected from the group consisting of polypeptides, small molecules,ribonucleic acids, and deoxyribonucleic acids.
 15. The method of claim11, wherein the method further comprises determining whether thecandidate compound increases survival of the motor neuron.
 16. Themethod of claim 11, the method further comprising: determining whetherthe candidate compound reduces a symptom of ALS in an animal model,wherein a candidate compound that reduces a symptom of ALS is acandidate compound to treat the symptom of ALS.
 17. The method of claim1, wherein the method further comprises administering to a subjectdiagnosed as having ALS a therapeutically effective amount of apotassium channel opener, thereby treating the ALS in the subject.